1. Field of the Invention
The present invention relates to a novel UDP-glycosyltransferase (uridine diphosphate glycosyltransferase) protein having glycosyltransfer activity for glucose linked by a glycosidic bond at the C-20 position of a PPD (protopanaxadiol)-type or PPT (protopanaxatriol)-type ginsenoside, and use thereof.
2. Description of the Related Art
Ginsenosides are glycosylated dammarene-type tetracyclic triterpenes, and they can be classified into three different groups based on their aglycone structure: protopanaxadiol (PPD)-type ginsenosides, protopanaxatriol (PPT)-type ginsenosides, and oleanolic acid-type ginsenosides. These three groups can be further classified based on the position and number of sugar moieties (aglycones) attached to the C-3, C-6, and C-20 positions of the rings by a glycosidic bond in the chemical structure. PPD and PPT also possess different hydroxylation patterns. PPD possesses —OH groups at the C-3, C-12, and C-20 positions, whereas PPT possesses —OH groups at the C-3, C-6, C-12, and C-20 positions. PPD and PPT can be glycosylated with glucose and other types of sugars to be converted into various ginsenosides. The representative PPD-type ginsenosides include ginsenoside Rb1, Rd, F2, Rg3, Rh2, CK (Compound K), Rb2, Rc, C-MC (Compound MC), and C-Y (Compound Y), and the representative PPT-type ginsenosides include Rg1, Rh1, F1, Rf, Re, and Rg2.
Meanwhile, the biosynthetic pathway of ginsenosides has only been partially identified. The ginsenoside biosynthesis is known to share the biosynthetic pathways with other triterpenes until oxidosqualene is synthesized by a series of condensation reactions of isopentenyl diphosphate and DMADP (dimethylallyl diphosphate) by the action of IPP isomerase (IPI), GPP synthase (GPS), FPP synthase (FPS), squalene synthase (SS), and squalene epoxidase (SE) (Ajikumar et al. Science, 330, 70-74. 2010; Ro et al. Nature, 440, 940-943. 2006; Sun et al. BMC genomics, 11, 262, 2010). Oxidosqualene is cyclized into dammarenediol-II by DS (dammarenediol-II synthase) which is a triterpene cyclase. Dammarenediol-II has hydroxyl groups at the C-3 and C-20 positions, and it is converted into PPD by hydroxylation of the C-12 position by a p450 enzyme, PPDS (protopanaxadiol synthase). PPDS can be also converted into PPT by hydroxylation at the C-6 position by another p450 enzyme, PPTS (protopanaxatriol synthase). PPD can be converted into various kinds of PPD-type ginsenosides by glycosylation at the C-3 and/or C-20 position(s), and PPT can be converted into various kinds of PPT-type ginsenosides by glycosylation at the C-6 and/or C-20 position(s).
UDP (Uridine diphosphate)-glycosyltransferase (UGT) is an enzyme that catalyzes the transfer of a sugar moiety from UDP-sugar to a wide range of metabolites such as hormones and secondary metabolites. Generally, UGT acts in the final step of biosynthetic pathway in order to increase solubility, stability, storage, bioactivity, or biological availability of metabolites. As recognized by a remarkable diversity of metabolites in plants, the genome of a plant possesses hundreds of different UGTs. For example, a plant model, Arabidopsis thaliana, contains 107 UGTs that belong to 14 different groups (Group A to Group N) based on the amino acid sequence. However, DS, PPDS, and PPTS have been reported as the enzymes involved in ginsenoside biosynthesis, but it has not been identified whether UGT is involved in the biosynthesis of ginsenosides. Thus, there is a need to investigate UGT that utilizes a ginsenoside as a substrate in order to produce a particular ginsenoside.
Further, different UGTs show substrate specificity towards both sugar donor and sugar acceptors. For example, UGT78D2 transfers glucose from UDP-glucose to the C-3 position of flavonol (kaempferol, quercetin) and anthocyanin (cyanidin) to produce flavonol 3-O-glucosides and cyanidin 3-O-glucoside, respectively. It seems that such glycosylation is essential for in vivo stability and storage of the compound. On the other hand, UGT89C1 transfers rhanmnose from UDP-rhanmnose to the C-7 position of flavonol-3-O-glucosides to produce flavonol-3-O-glucoside-7-O-rhamnoside. Likewise, UGT89C1 does not utilize UDP-glucose and anthocyanin-3-O-glucoside as a substrate, and it has different specificity towards UDP-sugars and acceptors from that of UGT78D2. As such, UGTs are different from each other in terms of substrate specificity and regioselectivity, and therefore, there is a need to investigate the substrate specificity and regioselectivity of different types of UGTs.
With this background, the present inventors have made many efforts to develop a novel UDP-glycosyltransferase with substrate specificity and regioselectivity which can be used for biosynthesis of a particular ginsenoside. As a result, the present inventors identified a novel glycosyltransferase GpUGT23 from Dolwoe (Gynostemma pentaphyllum), and they found that GpUGT23 has glycosyltransfer activity for glucose linked by a glycosidic bond at the C-20 position of PPD-type and PPT-type ginsenosides, and GpUGT23 can be used for the production of particular ginsenosides such as gypenoside LXXV, gypenoside XVII, Rb1, notoginsenoside U, notoginsenoside R3 or gluco-Re, thereby completing the present invention.